Abstract: Group I intron-derived ribozymes can be modified to perform a reaction called trans insertion-splicing (TIS) where the ribozyme binds two exogenous RNA substrates and inserts a sequence from one directly into the other. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and utilizes an ?G on the ribozyme and a 3?-G on the sequence being inserted. The internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. The reaction can also be performed without a first substrate, where the ribozyme is made with the insert sequence appended to its 3? end so as to perform a single substrate insertion targeted to any RNA sequence.